B cells get help in the antibody response by presenting processed antigen to helper T cells. We asked whether the antigen presenting B cell must induce T helper functions before receiving help, or whether B cell activation is a direct consequence of T cell antigen recognition on the B cell surface. Although antigen-dependent increases in B cell c-myc expression occur as early as two hours after conjugation, the B cell response depends on induction of a contact-dependent helper function in the T cell, which is inhibitable by cyclosporin A. Induction but not delivery of contact help is blocked by anti-class II MHC antibody, indicating that the delivery of T cell help is not Ag dependent or MHC restricted. Also, contact with activated helper T cells induces a different pattern of immediate early gene expression from signals transduced through the B cell antigen receptor. Egr-1 is rapidly upregulated in response to mitogenic signals induced by receptor crosslinking on murine B lymphocytes, and its expression closely correlates with B cell proliferation in several models of B cell activation and tolerance. We compared egr-1 expression during B cell stimulation with Fab'2 and IgG anti-Ig, since it is known that Fab'2 anti-Ig is mitogenic while IgG is not, due to a dominant inhibitory effect of crosslinking the B cell FcγRII to membrane Ig. While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in egr-1 expression, intact anti-Ig results in only small increases in egr-1 mRNA, comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig. However, when IL-4 is added as a comitogen to induce B cell proliferation with submitogenic concentrations of Fab'2 anti-Ig or IgG anti-Ig, no concomitant increases in egr-1 are observed. The regulation of egr-1 therefore, is similar to that of c-myc in this system, since neither correlates with IL-4 induced DNA synthesis.

Expression of germ-line α transcripts precedes class switching to IgA, and therefore study of the regulation of germ-line α RNA transcription is important for understanding the class switching process. Transforming growth factor β1 (TGFβ1) increases the transcription of the Ig constant region a gene and class switching to IgA in normal B cells and in the I.29μ B lymphoma cell line. The structure of germ-line α transcripts in I.29μ cells was analyzed by RNase protection and primer extension assays. Two initiation sites for germ-line α transcripts were identified 2 kb upstream to the α switch region. No TATA or Sp1 elements are found near the RNA initiation sites. The DNA segment located 5' to the initiation sites of germ-line α RNA can drive expression of a luciferase reporter gene when transiently transfected into I.29μ (subclone 22D) and A20.3 cell lines. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. In deletion and substitution mutation studies, an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line α promoter, but mutation of this motif does not diminish TGFβ1 inducibility. Induction by TGFβ1 requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemlyrepeated sequence 5' CACAG(G) CCAGAC 3' (termed Igα TGFβ-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats could confer TGFβ1 inducibility to a heterologous promoter. An additional copy of the TGFβ-RE was identified at -41/-30 and its deletion reduced the TGFβ1 response. Thus, tandem repeats of a novel TGFβ-RE are the positive regulatory elements for the TGFβ1 response. Gel mobility shift assays demonstrated specific binding to the TGFβ-RE by nuclear factors but the binding activity was not enhanced by TGFβ1. This study supports previously published evidence that TGFβ1 directs class switching to IgA through induction of germ-line Cα gene transcription.

Substance P (SP) is an undecapeptide whose functions are as varied as its locations. In the nervous system, it is thought to act as a neurotransmitter. In the peripheral vasculature, it has hypotensive effects and it causes contraction in the smooth muscle of the gut. In salivary gland, it is a potent secretagogue and it is how this effect is transduced that is the subject of this dissertation. Activation of the SP receptor in rat submaxillary gland by SP results in the hydrolysis of inositol phospholipids and the mobilization of intracellular Ca2+. These second messengers are then able to activate a pathway(s) which results in the secretion of electrolytes, water and macromolecules. The production of these second messengers, however, is thought to require the participation of a guanine nucleotide binding protein (G protein). The G protein that couples to the SP receptor (Gp), has not yet been identified. Although several investigators have recently reported the purification of G protein α subunits that are capable of activating phospholipase C, it is not known if they couple to receptors in order to activate phospholipase C. In an effort to learn more about the mechanisms of signal transduction by SP in salivary gland, the interactions of the SP receptor with G proteins were studied. In the first study, the question of whether the SP receptor functionally couples to a G protein was investigated. Alkaline treatment was used to deplete membranes containing SP receptors of endogenous G proteins. These membranes were not capable of binding SP with high affinity. High affinity binding capability was restored in those membranes, however, by reconstituting them with exogenous G proteins. Thus, it was concluded that that SP receptor agonist affinity is regulated by a G protein. It was also determined that the G proteins (a Go/Gi mixture) used to reconstitute the membranes may not be those that couple to the SP receptor in vivo, since the reconstituted Go/Gi mixture was inactivated by treatment with pertussis toxin, while Gp was not. The next study was undertaken in an effort to identify other G proteins that are able to interact with the SP receptor. G proteins were chromatographically purified from horse submaxillary gland membranes, and assayed for characteristics that could identify one or more G proteins as potential physiological couplers to the SP receptor. G proteins were identified in fractions by the ability to bind [35S]GTPγS. These GTP-binding proteins were further characterized by testing their susceptibility to ADP- ribosylation catalyzed by pertussis toxin and their ability to restore high affinity agonist binding in membranes containing the SP receptor, but no endogenous G proteins. In addition to identifying G proteins that are substrates for pertussis toxin-catalyzed ADP-ribosylation (e.g. Go and/or Gi), a GTP-binding protein was identified which possesses characteristics that are unlike those of the well-known G proteins, Go, Gi and Gs. This protein elutes from anion exchange resins at a high salt concentration, is not susceptible to ADP- ribosylation catalyzed by pertussis toxin, is able to reconstitute high affinity binding in G protein depleted rat submaxillary gland membranes and is not recognized by antibodies to Go, Gi, Gs or Gz. Finally, a direct characterization of the G protein coupled to the SP receptor in rat submaxillary gland was undertaken. Using photo-affinity labelling techniques in conjunction with chemical crosslinking techniques, a covalent 96 kDa SP receptor complex was identified. The generation of this 96 kDa complex was inhibited by a nonhydrolyzable analog of GTP, but not a nonhydrolyzable analog of ATP. Furthermore, the complex could not be produced in membranes that had been depleted of G proteins by alkaline treatment. Reversal of the chemical crosslink yielded only the 53 kDa SP receptor, showing that the protein crosslinking to the SP receptor possesses a molecular weight of about 43 kDa. This molecular weight is typical of G protein α subunits. It was concluded that the 96 kDa crosslinked receptor complex consisted of the SP receptor, the radioiodinated SP derivative and the α subunit of Gp. The studies show that the SP receptor may be coupled to a novel G protein, whose purification characteristics differ from those of the known G proteins. Although Gp has yet to be identified, comparisons of the results of these investigations with those of several recent articles in which the purification of G protein α subunits that are capable of stimulating phospholipase C is reported, suggests that Gp is similar, if not identical to those proteins. Furthermore, this dissertation describes a unique reconstitution system and crosslinking techniques which should prove useful in the identification of Gp, as well as in the study of other receptor-G protein interactions and perhaps, the reconstitution of the receptor-G protein-phospholipase C signal transduction pathway.

The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations. The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation. DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression.

mRNA decay is an important cellular process that regulates gene expression and is tightly linked to the process of translation. Many studies have illustrated the link between mRNA turnover and translation, indicating that mRNA decay is a cytoplasmic event. In order to investigate further the link between translation and turnover, seven mutants in translation initiation factors were analyzed for their effect on mRNA decay, including: i) three mutant alleles of the PRT1 gene (prt1-1, prt1-26 and prt1-63), which encodes a subunit of elF3; ii) sui1-1, which encodes the smallest subunit of elF3; iii) sui2-1, which encodes elF2; iv) GCN2c, which encodes the elF2 kinase, and v) cdc33-42, a mutant in the cap binding protein elF4E. The results demonstrate that the prt1-1 mutation results in stabilization of nonsense containing mRNAs without affecting the half-lives of most other mRNAs, a phenotype similar to a upf1Δ strain. To identify substrates for the nonsense-mediated mRNA decay pathway, mRNA differential display analysis was performed using RNA prepared from prt1-1, PRT1, upf1Δ and UPF1 strains. Although the abundance of the HHF2 mRNA is increased in the mutant strains the half-life is unaffected. However, the mRNA half-life of the transcriptional regulator SPT10 was increased in the mutant strains indicating the SPT10 transcript is a substrate for the nonsense-mediated mRNA decay pathway. Further characterization of the SPT10 transcript showed that it is a substrate for this pathway because the initiator AUG is present in a poor translation initiation context which results in aberrant translation initiation. Finally, several other mRNAs, predicted to be substrates for the pathway based on the leaky scanning model, were subsequently shown to decay through this pathway. These transcripts included the REV7, STE50, and UBP7 mRNAs. The results from these experiments lay the groundwork for addressing the potential regulatory role of the nonsense-mediated mRNA decay pathway.

In DNA immunization a plasmid expressing an antigen of interest is inoculated into an animal and antigen-specific humoral and cellular immune responses are raised. In this dissertation we sought to further our understanding of antibody responses raised following DNA inoculation. Specifically, we examined the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, the role of CD4+ and CD8+ T cells in the DNA-raised antibody response, the longevity of functional antigen expression, and the nature of the antigen presenting cell. In all of these studies mice were immunized with an influenza hemagglutinin-expressing plasmid and plasmid was delivered by either the gene gun or intramuscular routes of inoculation. To examine the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, responses raised in gene gun immunized mice were compared to responses raised in mice primed with an influenza infection. Antibody and antibody secreting cell (ASC) responses were analyzed at various times following immunization or sublethal infection for as long as 1.5 years. We found that the antibody response raised with a single gene gun immunization was similar in longevity to that raised in infection-primed mice. The long-term maintenance of the antibody response was associated with the localization of the majority of antibody secreting cells to the bone marrow. The kinetics of ASC bone marrow localization was 4-to-8 weeks slower in DNA-immunized than infection primed mice. This corresponded to a slower rise in the antibody response to plateau levels in DNA-immunized mice. We hypothesize that it is possible that the difference in kinetics may be linked to differences in the time course and dose of antigen expression following DNA immunization and a natural infection. Antibody and ASC responses were also compared following a challenge influenza virus infection. We found that DNA-immunized and infection-primed mice responded similarly in the acute post challenge phase with increases in antibody secreting cells in the mediastinal lymph nodes. While only DNA-immunized mice had post challenge increases in antibody, the antibody response remained 3-to-4 fold lower than post challenge responses in infection primed mice. We suggest that despite post challenge increases in these responses in DNA-immunized mice that the immune response raised with DNA immunization efficiently limited replication of the challenge virus and thus limited the post challenge antibody response. We also addressed the role that CD4+ and CD8+ T cells played in the ability to prime and boost the DNA-raised antibody response. To answer this question mice were in vivo depleted of CD4+ or CD8+ T cells for 3 weeks prior to through 2 weeks following DNA immunization or boost. Antibody responses were measured 4 and 8 weeks after DNA prime and 2 weeks after DNA boost. For both the gene gun and intramuscular routes of inoculation, the antibody response was independent of CD8+ T cells, but dependent on CD4+ T cells. The presence of CD4+ T cells was required at the time of DNA immunization, but not at the time of DNA boost. The absence of CD4+ T cells at the time of DNA delivery resulted in a four week delay in the appearance of antibody. Since influenza hemagglutinin has been characterized as a T-dependent antigen the requirement for CD4+ T cells at the time of DNA prime was not surprising, but the appearance of a delayed H1-specific antibody response suggested that DNA-expressed antigen had continued to be available to prime CD4+ T cells as they reappeared following the disappearance of depleting antibody. The independence of the antibody response on the presence of CD8+ T cells suggested that DNA-primed H1-specific CD8+ T cells did not limit the plateau level of response or the ability to boost a suboptimal response. The results from our CD4+ T cell depletion experiment suggested that DNA-expressed antigen continued to be available for an extended period of time following immunization. To examine the duration of functional antigen expression for raising an antibody response, mice lacking α/β T cells (TCR-/-) were immunized with DNA or immunized with hemagglutinin protein. Naive T cells from TCR+/+ mice were transferred into the immunized TCR-/- mice on various days post DNA or protein immunization. The results from these studies show that antigen is available to raise antibody longer following DNA immunization than following a protein immunization. This result is likely due to continued expression of plasmid DNA. We found differences in the longevity of antigen expression following gene gun and intramuscular routes of inoculation. For gene gun immunizations, not intramuscular immunizations, approximately 90% of functional antigen was lost within one week of immunization. We suggest that this is consistent with a role for antigen expression by transfected cells within the target site, the epidermis, which is largely lost by 1-2 weeks following gene gun immunization. We also found that following intramuscular immunization the dominant IgG isotype changed with time of TCR+/+ T cell transfer. By contrast, there was no change in the dominant isotype following gene gun immunizations. These results suggest that the factor(s) that contribute to the development of the Th1-bias seen following intramuscular DNA immunizations are lost early. To examine the nature of the antigen presenting cell following DNA immunization, dendritic cells were sorted from the inguinal lymph nodes and spleens of gene gun or intramuscularly immunized mice on various days following DNA delivery. The dendritic cell (CD11c+) and non-dendritic cell (CD11c-) populations were used in restimulation assays with H1-specific T cell clones. Despite similar titers of raised antibody in gene gun and intramuscularly immunized mice, H1-specific antigen presenting dendritic cells were isolated from the inguinal lymph nodes and spleens of gene gun, but not intramuscularly immunized mice. Antigen presentation by dendritic cells was detected for as long as 21 days following gene gun delivery. We hypothesize that the inability to detect dendritic cell presentation of antigen following intramuscular DNA delivery may be due to a more broad distribution of antigen presenting cells, different properties of antigen presenting cells, and/or the contribution of other non-dendritic cells to antigen presentation following intramuscular, but not gene gun, immunizations. We present our results within a model for the initiation and maintenance of DNA-raised antibody responses. Within this model our data specifically contribute to understanding the initiation and generation of the DNA-raised antibody response within lymphoid tissue and the maintenance of the DNA-raised antibody response.

Splicing of mRNA precursors (pre-mRNA) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use in splicing is not well understood. Several yeast splicing factors belong to DEAD/H box family of RNA-dependent ATPase, and are implicated in dynamic RNA structure rearrangement during spliceosome assembly. In mammals, however, such information is conspicuously lacking. In fact, none of the known mammalian splicing factors has characteristics for ATP hydrolysis. In an attempt to identify mammalian splicing factors involved in ATP usage, we have developed a novel approach to identify and purify spliceosomal ATP binding proteins. Six spliceosomal ATP binding proteins were found, one of them, SAFp56, was purified and microsequenced, and found to be a DEAD box protein containing unique DECD motif instead of the canonical DEAD motif. During the course of this work, a new functional region in U2AF65, an essential splicing factor required for U2 snRNP entry into the spliceosome, was defined. This information was used to clone a human U2AF65 associated protein (UAP). UAP and SAFp56 are identical. We refer to this protein as hUAP56 (human 56 kDa U2AF65 associated protein). We present evidence that hUAP56 is an essential splicing factor required for the U2 snRNP binding to pre-mRNA. Interestingly, UAP56 is recruited to pre-mRNA in a polypyrimidine tract bound U2AF65-dependent fashion. This result underscores a new function of U2AF65, and provides the first description of how a specific DEAD box protein is directed to a pre-mRNA splicing signal, and/or, to the proximity of its substrate at a particular stage. Like an authentic DEAD box protein. hUAP56 has ATP binding, RNA-stimulated ATPase, as well as RNA binding activity. A particularly novel result is that the ATPase activity of UAP56 is stimulated by U2AF65. This observation strongly suggests the role of UAP56 in ATP dependent mechanism during U2 snRNP binding to the pre-mRNA branchpoint, and implies that UAP56 may function through a distinct mechanism. We identify yeast UAP (yUAP), a highly conserved S. cerevisiae homologue of hUAP56. yUAP is essential for viability, can be functionally substituted for by hUAP56, and like its human counterpart, is an essential pre-mRNA splicing factor required for spliceosome assembly. Furthermore, we show that yUAP is required for formation of the branchpoint dependent commitment complex, the precursor for U2 snRNP addition. Site-directed mutagenesis revealed that all DEAD box protein consensus motifs are required for yUAP function. Interestingly, a strain harboring a yUAP mutant in which the DECD sequence, characteristic of UAP members, was changed to canonical sequence, is inviable. Our results demonstrate that UAP is structurally and functionally conserved from yeast to man. In conjunction with previous studies, we conclude that at least two DEAD box proteins, Prp5p and yUAP, are required for the U2 snRNP-branchpoint interaction.

In vivo infection with HIV-1 is typically characterized by progressive clinical and immunological deterioration associated with a decrease in CD4 T-cells. The mechanism of CD4 T-cell depletion in vivo and its role in HIV-1 pathogenesis and development of AIDS is not well understood. My research has focused on understanding the mechanism or mechanisms by which HIV-1 induces cell death. To address this aim, a panel of primary HIV-1 isolates were characterized in vitro for replication kinetics, syncytium formation, and cytopathic effects. From this panel two viral isolates, one from patient A and the other patient D, were selected for further evaluation. Patient A was asymptomatic with absolute CD4 cell count of 2302 at the time of virus isolation and remains so nine years later. Patient D was symptomatic with absolute CD4 cell count of 64 and has subsequently died from AIDS. Both of these patients maintained high viral load in vivo. When the viruses were examined in vitro, they also replicated to high titers. However, there were dramatic differences regarding the induction of cell death by HIV-1 isolates. Viruses obtained from patient A did not induce cell death although they replicated to high titers. In contrast, viruses obtained from patient D were extremely cytopathic in PBMCs with comparable viral replication. Therefore, viral replication alone does not predict the single-cell killing capacity of primary HIV-1 isolates. HIV-1 viruses isolated from an individual with normal CD4 T-cell numbers and an individual with CD4 T-cell depletion replicated to equivalent levels in primary CD4 T-cells. However, the virus isolated from the symptomatic individual induced cell death and the virus isolated from the asymptomatic individual was non-cytopathic in vitro. It is known that HIV-1 exists in the host as a group of related viruses known as quasispecies. This diversity allows the virus a broad spectrum of genotypes which result in multiple phenotypic properties. It follows then that a single viral isolate may contain a number of variants which differ in their ability to form syncytia, cell tropism, replication kinetics, as well as cytopathic potential. To address this, biological clones were obtained from each of the patients viral quasispecies and characterized for replication and cytopathicity. These clones, GC6 8-4 (isolated from patient A) and HC4 (isolated from patient D) maintained the same viral phenotype as the parental virus. In other words, HIV-1 biological clone GC6 8-4 was non-syncytium inducing (NSI) and non-cytopathic in vitro. In contrast, HIV-1 biological clone HC4 was syncytium inducing (SI) and cytopathic in vitro. It has been reported that the envelope gene of HIV-1 contains the major determinants of HIV-1 induced CD4 T-cell depletion (17). To understand what may be responsible for the differences in cytopathic behavior between the biological clones, GC6 8-4 and HC4 (42), I analyzed their envelope genes. Chimeric viruses were constructed by switching env regions from V2 through V3 of the biological clones with the corresponding region from the molecular clone NL4-3. These HIV-1 chimeric viruses exhibited similar replication kinetics as well as syncytium inducing abilities. The chimeric virus containing the env region of biological clone, GC6 8-4, was NCP in the single cell killing assay, while the chimeric virus containing the env region of biological clone, HC4, was CP in the single cell killing assay. These studies suggest the presence of a cytopathicity determinant which maps to the envelope region downstream from V2 and through V3 (Stu I at position 6822 to Nhe I at position 7250 based on NL4-3 sequence (101)).

Hepatitis C virus (HCV) is a positive strand RNA virus that is the leading cause of chronic hepatitis. HCV infections are an important health problem because >80% of patients become chronically infected and many develop chronic hepatitis. With approximately 400 million chronic HCV infections worldwide, understanding the pathogenesis of this disease is of critical importance in order to develop appropriate therapies and/or vaccine strategies. Strong proliferative and cytotoxic T cell responses that target multiple HCV proteins are detected in patients with self-limited infection. Conversely, HCV-specific T cell responses are minimal during acute infection in patients who become chronically infected. It is thought that the genetic diversity of HCV plays a crucial role in establishing persistence. Chronic viral hepatitis is characterized by infiltration of T lymphocytes in the liver, which are thought to play a pivotal role in disease progression. Although virus-specific T cells can be isolated from both peripheral blood and from liver biopsy samples of chronically infected patients, there appears to be a compartmentalization of HCV-specific T cells in the liver. However, the presence of virus-specific T cells is inefficient for viral clearance. Because HCV is known to be highly variable in sequence, the detailed characterization of the interaction of individual HCV-specific CTL clones with autologous viral sequences might be important for understanding the mechanisms by which HCV is able to establish a chronic infection. We isolated three intrahepatic CD8+ CTL clones from two individuals with chronic HCV infection and compared the recognition of prototype and autologous HCV sequences. These CTL recognized epitopes within the NS2 (amino acids 957-964) or NS3 (amino acids 1402-1410 and 1406-1415) proteins in the context of HLA B37, B8, or A2.1, respectively. The corresponding predominant autologous HCV sequences (SDWAANGL, ELAAKLVGL, ALRGMGLNAV, respectively) differed from the HCV-1 prototype sequences used for screening (RDWAHNGL, ELAAKLVAL, KLVALGINAV, respectively) at one to five residues. For each CTL clone, recognition of the autologous HCV sequence required significantly higher peptide concentrations than did recognition of the HCV-1 sequence; for two of the clones, recognition was minimal or absent at peptide concentrations as high as 25μM. When the HLA A2.1-restricted HCV NS3-specific T cell clone was analyzed further, we found that it was cross-reactive with peptide sequences from at least three other HCV strains. The clone recognized target cells loaded with synthetic peptides derived from sequences of genotype 1b; HCVTW (KLSALGIHAV), HCVJA (KLTGLGLNAV),and HCVBK (KLSGLGINAV). This HCV-specific T cell clone was also able to recognize target cells that were loaded with a peptide derived from an autologous protein, cellular retinoic acid binding protein I (CRABP I). When we generated HLA A2.1-restricted HCV NS3-specific T cell lines from the peripheral blood of two additional patients, almost one half of the cell lines could lyse target cells loaded with the CRABP I peptide. These data show that intrahepatic HCV-specific CD8+ CTL clones can be relatively inefficient at recognizing autologous viral epitopes and that some viral-specific CTL can recognize autoantigens in vitro. There is little information regarding the composition and stability of the liver-infiltrating T cell repertoire during chronic HCV infection. To address this issue, we used TCR complimentarity determining region 3 (CDR3) length analysis to examine the T lymphocytes in sequential biopsy samples from five individuals chronically infected with HCV. We found that although almost all TCRBV families were represented in the liver, 25-85% had skewed spectratype profiles, indicative of the presence of clonally expanded T cells. Further analysis using TCRBJ-primed run-off reactions revealed that the intrahepatic repertoires were not stable, as many expansions that existed in one biopsy sample were not detected in the other. Some expansions persisted, however, and sequencing of TCRBV-J transcripts identified CDR3 sequences that were maintained in two individuals for 10 or 45 months. Furthermore, although some expansions were found in the periphery, most were represented only in the liver. These data suggest that there is an evolution of the immune response during chronic HCV infection and that the response is largely concentrated in the liver of these individuals. Based on our observations regarding the function of intrahepatic HCV-specific CTL and the dynamics of the intrahepatic repertoire during chronic HCV infection, we propose a model in which the co-evolution of HCV quasispecies and HCV-specific T cells contribute to both viral persistence and immunopathology.

The organization and transcription of the gene for vasoactive intestinal polypeptide (VIP) in rats and mice was investigated using northern- and Southern-blot hybridizations, selective genomic cloning, Sl-nuclease protection assays, oligonucleotide-directed RNase H digestions, and genomic cloning by standard methods. The center of the rat VIP gene and the entire mouse gene were cloned and sequenced. Selective genomic cloning was used to isolate a strongly-hybridizing fragment of the rat VIP gene identified in Southern-blot hybridizations with an existing human VIP cDNA. This fragment contains separate exons encoding VIP and a closely related neuropeptide, peptide histidine-isoleucine (PHI-27). This organization is the same in the mouse gene, which bears a total of seven exons and a close similarity to the human gene for VIP. Although the arrangement of exons suggested that VIP transcripts could be subjected to differential splicing to alter the coding capacity of the final messages, no evidence was found for this possibility. Two bands were seen in northern-blot hybridizations, but exon-specific probes and Sl-protection experiments provided evidence that they differed not in their coding regions but in the extent of their 3'-untranslated ends. RNase H digestions targeted to specific portions of transcripts from the VIP gene were used to resolve the principal band, to demonstrate that it represented a single species of message with sequence from both the VIP and PHI exons. In the course of the characterization of the murine VIP gene, a new method was developed for generating subclones for DNA sequencing in M13 bacteriophage. The central feature of the partial deletion subcloning method is its employment of frequent-cutting restriction endonucleases to detach different extents of the insert from a construction. The viral construct is first linearized at a unique site between the insert and the site for hybridization of the M13 sequencing primer. The linearized construct is then subjected to partial digestion with different frequent-cutting restriction enzymes. Partially digested products are repaired and religated. Products with deletions from the insert now have the sequencing priming site religated to a portion of the insert that formerly had been distant. Most of the products with deletions in the viral genome are not viable and do not survive the procedure. Subclones are sorted from the pool of transfected products by sizing of single-stranded viral DNA by agarose gel electrophoresis. Selected subclones are subjected to a simple test for the presence of the sequencing priming site. With this method and its associated tests, a variety of restriction enzymes can be used to generate a spectrum of deletion subclones for sequencing. In a simple trial of this method with an unknown 3.3 kilobasepair cDNA, a set of subclones was generated to allow sequencing to span the cDNA in one direction.